Primary structure of single-chain pro-urokinase

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Chemistry

Background:

• Single-chain pro-urokinase (pro-UK) is an inactive precursor of human urokinase.
• Activation involves conversion to a two-chain form by plasmin.
• Understanding the precise activation mechanism is crucial for thrombolytic therapy.

Purpose of the Study:

• To elucidate the molecular mechanism of single-chain pro-urokinase activation.
• To identify the specific cleavage site responsible for converting pro-UK to active UK.
• To correlate structural changes with functional alterations.

Main Methods:

• Isolation of single-chain pro-urokinase from human kidney cell culture.
• Chemical (CNBr) and proteolytic degradation.
• Peptide separation using reverse-phase high-performance liquid chromatography (RP-HPLC).
• Automated amino acid sequence analysis.

Main Results:

• Determined the amino acid sequence of 85% of residues in 17 peptides.
• Confirmed sequence consistency with previously reported A and B chains of human urokinase.
• Identified Lysine at position 158 as the linkage point for the two chains.
• Demonstrated plasmin cleavage at the Lys158-Ile159 bond, yielding active two-chain urokinase.

Conclusions:

• The Lys158-Ile159 bond cleavage is the critical step in single-chain pro-urokinase activation.
• This cleavage induces a conformational change, activating enzyme function.
• Activation significantly reduces the affinity of urokinase for fibrin.